Cancer involves the induction of uncontrolled DNA replication and mitosis in cells, as well as processes that ensure cells evade cell death or senescence and survive in specific tissue micro-environments. Project 1 has been a leader in studying the mechanisms and control of inheritance of the human genome and has identified many of the key proteins that are involved in DNA synthesis at the replication fork and other proteins that are involved in the initiation of DNA replication. In the proposed studies. Project 1 will continue to focus on how the initiation of DNA replication is controlled in human cells and how this process goes awry in tumor cells. Specific Aim 1 will focus on how the origins of DNA replication are marked in chromosomes so that they can form pre-replicative complexes during exit from mitosis or during Gl phase, thereby enabling the initiation of DNA replication in S phase of the cell division cycle. Since the Origin Recognition Complex, particularly its largest subunit Orel is loaded onto chromosomes beginning in prophase of mitosis and Orel is the most stably bound chromatin protein, the locations within the human genome for Orel binding will be determined. In addition, proteins that dynamically interact with ORC during M and G1 phases will be determined. The cell cycle regulators Cyclin E-CDK2 and Cyclin A-CDK2, the former often over-active in breast cancer, are controlled by direct interactions with Orel and Cdc6 and how these interactions influence the initiation of DNA replication and centriole duplication in centrosomes will be investigated. Recent evidence has emerged that ORC subunits play a critical role at kinetochores that bind microtubule spindles for congression of chromosomes prior to their segregation. In Specific Aim 2, interactions between the Orc2 and Orc3 subunits of ORC and the Spindle Assembly Checkpoint kinase BubRI will be investigated, as will the role of the ORC subunits in maintenance of stable spindle attachment during the metaphase to anaphase transition. In Specific Aim 3, the control of DNA replication by the DEAD-box RNA helicase DDX5 and its interaction with the transcription factor E2F1 will be studied. DDX5 is amplified in the genome of cells in 25% of human breast cancers and it is these cells that display selective sensitivity to inhibition of DDX5 protein levels. How DDX5 influences E2F1-driven expression of DNA replication genes in the Gl phase of the cell cycle will be investigated. Project 1 will also investigate how tumor cells with amplified copy number of DDX5 become addicted to its continued expression, in contrast to normal, non-tumor cells and many other cancer cells. Finally, the additive effects on inhibition of tumors cell proliferation with the combination of DDX5 depletion and Trastuzumab (Herceptin) treatment will be examined.